Alkaline phosphatase (APase) is an enzyme which catalyzes the hydrolysis of an ester bond of terminal phosphate groups (PO.sub.4.sup.-). This reaction is useful as a research tool particularly for radioactive end-labeling of nucleic acids using T4 polynucleotide kinase to study nucleic acid structure and function.
In the procedure for radioactive end-labeling, the substrate, such as DNA, RNA or an oligonucleotide, is first dephosphorylated with APase prior to the labeling step. In the next step, the APase activity must then be eliminated to avoid both degradation of ATP and loss of label from the nucleotide substrates. Finally, polynucleotide kinase (PNK) is used to catalyze the phosphorylation of the substrate, wherein the phosphate donor is typically a gamma-P.sup.32 ATP. As noted above, residual APase activity in the reaction vessel during or subsequent to the phosphorylation step, can result in a loss of label from the ATP and polynucleotide substrate, thereby destroying the results of the radioactive end-labeling reaction.
Most researchers are using APase isolated from the microorganism E. coli, which is available commercially, to cleave the terminal phosphates from the nucleotide substrate. The disadvantage of this enzyme is its great stability, especially its heat stability, which makes inactivation of the APase particularly difficult.
Severaly methods are currently employed to remove or inactivate E. coli APase from the phosphorylation reaction vessel prior to the labeling step. The most effective method is phenol extraction; however, this method is time-consuming, results in poor recovery of nucleic acids and is inappropriate for processing large numbers of samples. Methods of treatment with NaOH, HCl, boiling, or nitriliatriacetic acid are not suitable because the APase is not completely inactivated thereby. One other method is the use of inorganic phosphate (Pi) to inhibit APase activity. This method is disadvantageous in that Pi also inhibits polynucleotide kinase activity, so that the phosphate labeling of highly structured polynucleotide substrate, such as DNA or RNA, is also greatly reduced. The general problem with the above methods is that E. coli APase is highly stable making removal or inactivation thereof difficult.
One solution to the above-noted problems of the stability of E. coli APase in end-labeling experiments is to use heat sensitive APase. Some heat sensitive APases are disclosed in Alkaline Phosphatase, R. B. McComb, et. al. (1979), Plenum Press, N.Y. p. 404 in "Table of Thermal Denaturation Rates of Selected Microbial Alkaline Phosphatases." As stated therein, the APases which are most heat sensitive include Bacillus megaterium with a half-life of 4 minutes at 55.degree. C., and Sacharomyces cerevisiae with a half-life of 2.5 minutes at 60.degree. C. However, the report of these temperature sensitive APases does not disclose what treatment is necessary for complete APase inactivation, and there is no necessarily direct relationship between half-life (50% inactivation of enzyme activity) and complete inactivation of the enzyme. Moreover, effective phosphorylation of the polynucleotide substrate can only occur if the APase is completely inactivated.
One particularly significant problem with using temperature treatment to inactivate APase is that double stranded polynucleotides are somewhat heat labile. This problem is caused by the fact that double stranded DNA, RNA and oligonucleotides are held together by hydrogen bonding which can be broken at elevated temperatures. Shorter polynucleotide double stranded chains and those polynucleotides containing a large percentage of adenine-thymine base pairs are examples of particularly temperature sensitive polynucleotides. In fact, double stranded DNA can be completely denatured by heat treatment at 65.degree. C. Thus, it is preferable for radioactive end-labeling procedures, where the integrity of the polynucleotide substrate may be important that the temperature of heat treatment of the polynucleotide substrate be as low as possible, and the duration of such treatment be short. The present invention obviates the need for higher elevated temperatures or other extreme conditions for removing or inactivating APase.